The field of the invention is heparin-binding growth factors (HBGFs) This work was supported in part by a grant from the U.S. government, which has certain rights in the invention.
The HBGFs are a family of mammalian growth factors related by sequence homology, receptor affinity, and, as their name implies, the ability to bind heparin. Heparin is a glycosaminoglycan (GAG) with anticoagulant activity commercially isolated from mammalian tissues as a heterogeneous mixture of variably sulfated polysaccharide chains (molecular weight 6-30 kDa) composed of repeating units of D-glucosamine and either L-iduronic acid or D-glucuronic acid. Binding to heparin has been shown to stabilize at lease some of the HBGFs against heat denaturation and proteolytic degradation (Gospodarowicz and Chen, J. Cell Physiol. 128:475-484, 1986; Saskela et al., J. Cell. Biol. 107:743-751, 1988).
Members of the HBGF family which have been identified to date include acidic fibroblast growth factor (aFGF), basic FGF (bFGF), int2 and HST (both of which are considered to be oncogenes expressed at abnormally high rates in certain cancers), K-FGF (first seen in a Kaposi's sarcoma), FGF-5, FGF-6, and keratinocyte growth factor (KGF) (Rubin et al., Proc. Natl. Acad. Sci. USA 86:802-806, 1989; Folkman and Klagsbrun, Science 235:442-447, 1987; Klagsbrun, Progress in Growth Factor Research 1:207-235, 1989). The HBGFs stimulate proliferation, migration and differentiation of cells of mesenchymal and neuroectodermal origin. bFGF, one of the more thoroughly studied HBGFs, participates as an autocrine modulator of cell growth and transformation and is a potent angiogenic factor abundant in normal and malignantly transformed cells (Rifkin and Moscatelli, J Cell. Biol. 109:1-6, 1989; Yayon and Klagsbrun, Proc. Natl Acad. Sci. USA 87:5346-5350, 1990). The strong affinity of bFGF for heparin has greatly facilitated the purification and characterization of this growth factor (Shing et al., Science 223:1296-1299, 1984; Klagsbrun and Shing, Proc. Natl. Acad. Sci USA 82:805-809, 1985). Heparin is also a potent modulator of the biological activity of bFGF and aFGF: e.g., heparin has been found to potentiate the mitogenic effect of aFGF on endothelial cells (Thornton et al., Science 222:623-625, 1983). Protamine, a protein that binds avidly to heparin, inhibits the ability of heparin to stimulate endothelial cell migration (Azizkhan et al., J. Exp. Med. 152:931-944, 1980) and inhibits angiogenesis associated with embryogenesis and inflammation (Taylor and Folkman, Nature 297:307-312, 1982).
The biological response of cells to the HBGFs is mediated through specific, high-affinity (Kd=2-20.times.10.sup.-11 M) cell surface receptors which possess intrinsic tyrosine kinase activity and are phosphorylated upon binding of an HBGF (Coughlin et al., J. Biol. Chem. 263:988-933, 1988). Several closely related glycoproteins from various species have been denominated FGF receptors: these include the chicken FGF receptor (CEK) and the protein encoded by the human cDNA clone flg, as well as others (Lee et al., Science 245:57-60, 1989; Ruta et al., Oncogene 5:635-643, 1989; Kornbluth et al., Mol. and Cell. Biol. 8:5541-5544, 1988; Pasquale et al., Proc. Natl Acad. Sci. USA 86:5449-5433, 1990; Safran et al., Oncogene 5:635-643, 1990). A lower-affinity (Kd=10.sup.-7 -10.sup.-9 M), large-capacity class of binding sites has also been identified (Moscatelli, J. Cell Biol. 131:123-130, 1987). These low-affinity binding sites are heparan sulfate proteoglycans (HSPGs; a class of protein-linked polysaccharides with a sugar structure similar to heparin, but having more N-acetyl groups and fewer O- and N-sulfate groups than does heparin) found on the cell surface (Moscatelli, J. Cell Biol. 107:753-759, 1988) and in the extra-cellular matrix (Vlodavsky et al., Proc. Natl. Acad. Sci. USA 84:2292-2296, 1987). bFGF can be released from these low-affinity binding sites by an excess of heparin or by enzymatic digestion with heparinases, but not with closely related GAGs such as chondroitin sulfate or by enzymes such as chondroitinase or hyaluronidase (Moscatelli, J. Cell Biol. 107:753-759, 1988; Bashkin et al., Biochem. 28:1737-1743, 1989). Recently, it has been shown that herpes simplex viruses, which are capable of binding to cell surface HSPG (WuDunn and Spear, J. Virol. 63:52-58, 1989), use the high-affinity FGF receptor as a portal of entry into cells (Kaner et al., Science 248:1410-1413, 1990).